Typically, production process of peptide or protein of interest by host cells, or molecular cloning, requires the use of one or more replicon sequence(s), such as plasmids carrying one or more gene(s) encoding the protein(s) of interest.
Cells in bioreactor present usually a high heterogeneity. It has been demonstrated that the growth of plasmid bearing cells, especially bacterial cells, is significantly reduced compared to plasmid free host cells. Indeed, peptide or protein production coming from gene of interest overproduction represents a significant burden on the metabolism of the cell.
Therefore, it is necessary to select cells that keep in their (chromosomal or extrachromosomal) genome the gene of interest.
This selection is efficiently done by the use of a selectable marker, such as antibiotic resistance gene or pair of poison and/or antidote genes.
Poison (or toxin) and antidote (or antitoxin) genes are sequences naturally found in plasmids, chromosomes and bacteriophages.
A poison gene, or a nucleotide sequence of such a gene, will code for a stable peptide or protein whereas its corresponding antidote gene can code for another peptide, protein or an RNA, stable or unstable, that may neutralize the poison peptide or protein either transcriptionally or post-transcriptionally or post-translationally or via protein-protein or RNA-protein interaction(s).
Because it represents a burden to cell metabolism, bacteria will not always produce the recombinant peptide or proteins efficiently. Indeed, it is possible that non desired genetic modifications are generated in the protein coding sequence, for instance point mutations (nucleotide(s) substitutions), deletions or additions of one or more nucleotide(s) in the recombinant nucleotide(s) sequence(s) of interest will occur and will generate stop codons, frameshifts and/or deletion or insertion in the corresponding amino acid sequence(s) and maintain the production of inactive, mutated or truncated peptides or proteins. At the level of transcription, errors can also occur such as nucleotide(s) substitution(s), deletions or additions of one or more nucleotide(s), or premature arrest of transcription, resulting in aberrant messenger RNA, which produce inactive, mutated or truncated peptides or proteins. Even in the absence of errors at the genetic or messenger RNA levels, errors can occur during translation such as frameshifts or premature arrest of translation, which also produce inactive, mutated or truncated peptides or proteins. In addition, mutations can occur elsewhere in the genome (including chromosomal and extra-chromosomal elements), which can directly or indirectly affect the yield and quality of the recombinant protein or peptide, such as mutations affecting the promoter or operator region of the gene encoding the protein or peptide, or mutations affecting the expression of components of the transcriptional or translational machineries.
In addition, due to high heterogeneity in bioreactors, some cells show no or low recombinant peptide or protein production.
Therefore, it still exists a need to improve methods and systems to guarantee better quality of recombinant peptides or proteins, particularly of full-length recombinant peptides or proteins showing the expected primary amino acid sequence, and to guarantee homogeneous producing-cell population, possibly present in a bioreactor.